Background
Autism spectrum disorder (ASD) stands as a neurodevelopmental disorder characterized by intricate hurdles in social communication and interaction, alongside a display of constrained interests and repetitive behaviors [
1,
2]. Under the umbrella term of autism spectrum disorder lie several closely linked conditions, encompassing Asperger’s syndrome, pervasive developmental disorders not otherwise specified (PDD-NOS), and childhood disintegrative disorder [
3‐
5]. According to present epidemiological investigations, the estimated prevalence rate of ASD hovers around 1 in 100 children; however, certain regions within the United States report significantly higher rates, reaching as steep as 1 in 44 [
6]. This variation in prevalence rates might be attributed to differing diagnostic criteria, variances in healthcare accessibility, and disparities in ASD awareness across specific regions. Amidst this fluctuation, the undeniable reality emerges that ASD presents a substantial public health apprehension demanding ongoing investigation and focus. Through an in-depth grasp of the factors underpinning ASD prevalence, we can more effectively recognize and bolster individuals bearing ASD and their families, while simultaneously forging potent prevention and intervention strategies. Notably, in China, the count of children affected by ASD ascends into the millions, and this figure persists in its upward trajectory. These children routinely encounter obstacles in communication, language acquisition, self-care, and the pursuit of knowledge. Furthermore, they may incline toward solitary behaviors. In severe instances, the potential exists for them to exhibit self-injurious or aggressive tendencies [
7].
The etiology of ASD remains to be comprehensively deciphered. A myriad of elements, encompassing genetics, prenatal and perinatal circumstances, environmental impacts, social determinants, and family interplay, are all conceivably implicated [
7]. Despite the array of interventions accessible for ASD, encompassing rehabilitative training, psychological therapy, and medication, these methods frequently prove insufficient in attaining the sought-after curative results. These constraints underscore the necessity for extended research endeavors and pioneering approaches in treating ASD [
4,
8].
The human gastrointestinal tract harbors a diverse and extensive microbiota. With a population ranging from 10
13 to 10
14 microorganisms, the gut microbiota possesses a genome over 100 times larger than of humans [
9], playing pivotal roles in the host’s well-being. It ferments indigestible food components into absorbable metabolites, synthesizes essential vitamins, eliminates harmful toxins, resists pathogens, bolsters the intestinal barrier, and regulates the immune system [
10]. Extensive research underscores the crucial engagement of the gut microbiota in both the development and operation of the host’s adaptive immune system, as well as in shaping the central nervous system [
11‐
13]. Therefore, gut microbiota dysbiosis can lead to various disorders affecting the gastrointestinal, immune, and neurodevelopmental systems, including ASD [
14]. Clinical observations reveal that children with ASD frequently experience chronic gastrointestinal issues like constipation and diarrhea, with constipation being the most frequently noted [
15]. Importantly, the severity of gastrointestinal symptoms in ASD-affected children correlates directly with the extent of their condition. Those grappling with persistent gastrointestinal problems tend to exhibit deficient social skills and heightened difficulty in emotional regulation. Encouragingly, studies demonstrate that enhancements in these gastrointestinal symptoms correspond to diminished severity of social and emotional difficulties in children with ASD [
16].
While numerous studies have underscored distinctions in gut microbiota between individuals with ASD and their healthy counterparts, the quest for advantageous bacteria or viable metabolic products for clinically treating ASD remains unfruitful. This dearth of discovery could stem partly from the gut microbiota’s limited contribution to the onset and progression of ASD. Besides, disparities in culture, diet, environment, and customs across global regions and populations can substantially influence gut microbiota variations among individuals. Therefore, by broadening the spectrum of screened individuals, the probability of identifying microbiota associated with ASD is heightened. In this study, we aimed to analyze the gut microbiota composition by collecting fecal samples from both healthy individuals and those with ASD in the southeastern coastal region of China. Through 16 S rRNA gene sequencing, we strive to furnish an intricate depiction of gut microbiota profiles in individuals with ASD and potentially uncover novel microbiota associated with this condition.
Discussion
The emergence of ASD during childhood is closely linked to the intricate world of gut microbiota. Nonetheless, owing to the intricate interplay of factors like culture, dietary habits, environment, and customs, substantial disparities in gut microbial communities exist among individuals inhabiting diverse geographical regions [
21,
22]. Presently, there is a lack of information regarding the distribution of gut microbiota among children with ASD in the southern expanse of China. Consequently, the objective of this study is to bridge this knowledge gap by collecting fecal samples from both healthy children and those diagnosed with ASD residing in the southeastern coastal area of China, specifically Fujian Province. The 16 S rRNA gene sequencing was utilized to analyze gut microbiota in children with ASD. As a demonstrative example, Fig.
1C presents the AS30 sample, exhibiting a significantly high abundance of
c_Fusobacteria. Likewise, Fig.
1D highlights the AS9 sample, indicating a notable increase in
o_lactobacillales. Consistent with previous research, it was observed that children diagnosed with ASD exhibit an atypical gut microbial composition in comparison to their typically developing peers [
23‐
25]. Certain pathogenic bacteria were found to be present at higher abundance in gut microbiota of children with ASD than in normal children, such as
Clostridia, Bacteroidetes, and
Desulfovibrio [
26]. These results provide further evidence that gut microbial composition of children with ASD significantly differs from that of healthy children.
The taxa exhibiting significant abundance differences between the ASD and control cohorts were identified. In the control group,
g_Acinetobacter,
g_Flavonifractor,
g_Prevotellaceae_NK3B31_group, and
g_UBA1819 displayed higher abundance compared to the ASD group. Conversely, the ASD group exhibited higher abundance of
g_Ruminococcus_1,
g_Streptococcus, and
g_Lachnospiraceae_NC2004_group when compared to the control group. These observed changes in differential genera between the two groups align with the findings of several previous studies. For instance, Zhang et al. observed the proportion of Firmicutes/Bacteroidetes in ASD was significantly increased, accompanied by pronounced enrichment of
Lachnospiraceae and
Ruminococcaceae families. In their study, eighteen genera, including
Ruminococcus,
Blautia, and
Holdemanella were notably elevated in the ASD cohort [
27]. Additionally, Li et al. employed 16 S rRNA gene sequencing to analyze stool samples from 59 mother-child pairs with ASD children and 30 matched pairs of healthy children, revealing distinctive bacterial biomarkers like
Alcaligenaceae and
Acinetobacter within the ASD group [
28]. These findings collectively underscore the variability of microbial community structures across different cohorts and regions, highlighting the need for diverse regional sampling to pinpoint region-specific bacteria potentially linked to disease.
Given that gut microbiota structure of children with ASD undergoes significant changes, researchers have delved into the potential of modulating gut microbiota as a therapeutic measure for this condition. In a previous study, 18 children with ASD underwent a regimen of Fecal Microbiota Transplantation (FMT) spanning 18 weeks. Following an 8-week period of FMT treatment, marked enhancements were observed in the scores of the ASD Diagnostic Observation Schedule (ADOS), the ABC, and the Social Responsiveness Scale (SRS) in children with ASD, in comparison to their pre-treatment scores. Additionally, the Gastrointestinal Symptom Rating Scale (GSRS) revealed significant improvement in gastrointestinal symptoms, including constipation, diarrhea, indigestion, and abdominal pain [
15]. successful engraftment of certain donor microbial communities in the gut, leading to heightened diversity of the gut microbiota. These findings suggest the potential efficacy of FMT as a short-term intervention for alleviating both social and gastrointestinal symptoms in children with ASD. Furthermore, these outcomes hint at the possibility of FMT serving as a viable long-term approach for ASD treatment [
15]. Given the variances in microbial community structures across populations and regions, accompanied by differing bacterial species accountable for these distinctions, it becomes apparent that personalized microbial FMT might represent a feasible avenue. Recent investigations have illustrated that aligning the gut microbiota of fecal donors with FMT recipients can effectively address conditions like ulcerative colitis (UC). This matching paradigm offers the prospect of selecting well-suited fecal donors for patients with UC [
29,
30]. Hence, a promising direction for improving treatment efficacy in children with ASD could be applying the donor-recipient matching FMT strategy.
The clinical manifestations of childhood ASD are varied, and aside from mental symptoms, gastrointestinal symptoms also play a significant role in diagnosis [
15,
16]. As previously observed, there is a noticeable alteration in the structure of intestinal microbiota in individuals with ASD; however, the precise causal relationship between the two remains incompletely comprehended. Recent studies have elucidated the crucial role of bacterial metabolites in regulating diverse facets of the host’s metabolism, including but not limited to appetite regulation, glucose homeostasis, energy expenditure, and immune response. These metabolites serve as intermediary agents through which the gut microbiota exerts its effects on the host [
31,
32]. Gut microbiota dysbiosis causes significant alterations in the metabolomic profile, characterized by elevated levels of short-chain fatty acids (SCFAs) such as propionic and butyric acids. These changes can be attributed to the substantial proliferation of bacterial genera
Clostridia, Bacteroidetes, and
Desulfovibrio, which have been previously linked to patients with ASD [
33]. Particularly noteworthy is the augmented presence of Clostridia, known producers of propionic acid, which has been observed to be significantly higher in the fecal samples of ASD children suffering from constipation. Moreover, the heightened levels of propionic acid in fecal samples align with this, suggesting a potential link between constipation and the abundance of propionic acid-producing bacteria [
34,
35]. In our study, we utilized bioinformatics analysis to confirm the changes in metabolic pathways that corresponded to different microbial communities in local ASD patients. Our study revealed a significant reduction in alpha-linolenic acid metabolism, paving the way for deeper exploration into the intricate molecular mechanisms that underpin the impact of gut microbiota on the development of ASD.
Our study possesses several limitations. Firstly, our analysis focused on bacterial profiles derived from the 16 S rRNA gene. Consequently, this examination omitted the finer strain-level distinctions within gut bacterial taxa, as well as other microbial components such as the fungal microbiota and virome. Secondly, conducting fecal collection at different time frames and employing longitudinal sampling is a valuable approach to comprehensively study the gut microbiota’s dynamics, understand its responses to various factors, and uncover potential associations with health outcomes. It helps mitigate the inherent variability of the microbiota and provides a more accurate representation of its behavior over time [
36,
37]. By analyzing a disease-free Swedish population over a year, utilizing both whole-genome metagenomic and 16 S rRNA gene sequencing, study by Olsson LM et al. underscores the significance of temporal dynamics in the gut microbiota, which can impede the identification of consistent microbial markers associated with health [
36]. Moreover, longitudinal analysis about ASD suggests that variations in lethargy/social withdrawal levels among individuals at different time points exhibited a correlation with alterations in gut microbiome composition. Additionally, a decline in gut microbiome diversity was found to be linked to an exacerbation of inappropriate speech between the aforementioned time points [
37]. The composition of the gut microbiota is not fixed and undergoes ongoing adaptive modifications. Paradoxically, He et al. demonstrated the stability of gut microbial communities within a specific timeframe [
17]. Frost et al. observed a remarkable stability in the overall structure of fecal microbiome communities over time at a population level [
38]. In the current analyses, we did not employ a longitudinal sampling approach to evaluate the dynamic changes in gut microbiota over time. Our assessment of the gut microbiota in ASD children was constrained to data from a single sampling time point. Hence, to gain a more comprehensive understanding of the dynamic nature of gut flora in ASD patients, we intend to establish multiple sampling time points and implement diverse intervention protocols in our future investigations.
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